Chimeric adenoviruses for use in cancer treatment

ABSTRACT

The present invention relates to oncolytic adenoviruses having therapeutic applications. Recombinant chimeric adenoviruses, and methods to produce them are provided. The chimeric adenoviruses of the invention comprise nucleic acid sequences derived from adenoviral serotypes classified within the subgroups B through F and demonstrate an enhanced therapeutic index.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/574,851, filed on May 26, 2004, which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention described herein relates generally to the field ofmolecular biology, and more specifically to oncolytic adenoviruseshaving therapeutic applications.

BACKGROUND OF THE INVENTION

Cancer is a leading cause of death in the United States and elsewhere.Depending on the type of cancer, it is typically treated with surgery,chemotherapy, and/or radiation. These treatments often fail, and it isclear that new therapies are necessary, to be used alone or incombination with classical techniques.

One approach has been the use of adenoviruses, either alone or asvectors able to deliver anti-cancer therapeutic proteins to tumor cells.Adenoviruses are non-enveloped icosohedral double-stranded DNA viruseswith a linear genome of approximately 36 kilobase pairs. Each end of theviral genome has a short sequence known as the inverted terminal repeat(or ITR), which is required for viral replication. All human adenovirusgenomes examined to date have the same general organization; that is,the genes encoding specific functions are located at the same positionon the viral genome. The viral genome contains five early transcriptionunits (E1A, E1B, E2, E3, and E4), two delayed early units (IX and Iva2),and one late unit (major late) that is processed to generate fivefamilies of late mRNAs (L1-L5). Proteins encoded by the early genes areinvolved in replication, whereas the late genes encode viral structuralproteins. Portions of the viral genome can be readily substituted withDNA of foreign origin and recombinant adenoviruses are structurallystable, properties that make these viruses potentially useful for genetherapy (see Jolly, D. (1994) Cancer Gene Therapy 1:51-64).

Currently, the research efforts to produce clinically useful adenoviraltherapy have focused on the adenoviral serotype, Ad5. The genetics ofthis human adenovirus are well-characterized and systems are welldescribed for its molecular manipulation. High capacity productionmethods have been developed to support clinical applications, and someclinical experience with the agent is available. See, Jolly, D. (1994)Cancer Gene Therapy, 1:51-64. Research related to the use of humanadenoviruses (Ad) in cancer treatment has focused on the development ofAd5-based adenoviruses that have a higher potency in, or arepreferentially targeted to, specific tumor cell types and there exists aneed for generation of more potent oncolytic viruses if adenoviraltherapy is to find practical application in a clinical setting.

Ad5 is only one of 51 currently known adenoviral serotypes, which areclassified into subgroups A-F, based on various attributes includingtheir hemagglutination properties ((see, Shenk, “Adenoviridae: TheViruses and Their Replication,” in Fields Virology, Vol. 2, FourthEdition, Knipe, ea., Lippincott, Williams & Wilkins, pp. 2265-2267(2001)). These serotypes differ at a variety of levels, e.g. pathologyin humans and rodents, cell receptors used for attachment, but thesedifferences have been largely ignored as potential means to develop morepotent oncolytic adenoviruses (with the exception of fiber alterations,see Stevenson et al. (1997) J. Virol. 71:4782-4790; Krasnykh et al.(1996) J. Virol. 70:6839-6846; Wickham et al. (1997) J. Virol.71:8221-8229; Legrand et al. (2002) Curr. Gene Ther. 2:323-329; Barnettet al. (2002) Biochim. Biophys. Acta 1-3:1-14; U.S. patent applicationSer. No. 2003/0017138).

Exploitation of differences among adenoviral serotypes may provide asource of more effective adenoviral-based therapeutics, using noveladenoviruses with increased selectivity and potency. There is a need forsuch improved adenoviral-based therapies.

SUMMARY OF THE INVENTION

The present invention provides novel chimeric adenoviruses, or variantsor derivatives thereof, useful for viral-based therapy. In particular,the invention provides for chimeric adenoviruses, or variants orderivatives thereof, having a genome comprising an E2B region

-   -   wherein said E2B region comprises a nucleic acid sequence        derived from a first adenoviral serotype and a nucleic acid        sequence derived from a second adenoviral serotype;    -   wherein said first and second adenoviral serotypes are each        selected from the adenoviral subgroups B, C, D, E, or F and are        distinct from each other; and    -   wherein said chimeric adenovirus is oncolytic and demonstrates        an enhanced therapeutic index for a tumor cell.

In one embodiment, the chimeric adenovirus further comprises regionsencoding fiber, hexon, and penton proteins, wherein the nucleic acidsencoding said proteins are all from the same adenoviral serotype. Inanother embodiment, the chimeric adenovirus of the invention comprises amodified E3 or E4 region.

In another embodiment, the chimeric adenovirus demonstrates an enhancedtherapeutic index in a colon, breast, pancreas, lung, prostate, ovarianor hemopoietic tumor cell. In a particularly preferred embodiment, thechimeric adenovirus displays an enhanced therapeutic index in colontumor cells.

In a preferred embodiment, the E2B region of the chimeric adenoviruscomprises SEQ ID NO: 3. In a particularly preferred embodiment, thechimeric adenovirus comprises SEQ ID NO: 1.

The present invention provides for a recombinant chimeric adenovirus, ora variant or derivative thereof, having a genome comprising an E2Bregion

-   -   wherein said E2B region comprises a nucleic acid sequences        derived from a first adenoviral serotype and a nucleic acid        second derived from a second adenoviral serotype;    -   wherein said first and second adenoviral serotypes are each        selected from the adenoviral subgroups B, C, D, E, or F and are        distinct from each other;    -   wherein said chimeric adenovirus is oncolytic and demonstrates        an enhanced therapeutic index for a tumor cell; and    -   wherein said chimeric adenovirus has been rendered replication        deficient through deletion of one or more adenoviral regions        encoding proteins involved in adenoviral replication selected        from the group consisting of E1, E2, E3 or E4.

In one embodiment, the chimeric adenovirus of the invention furthercomprises a heterologous gene that encodes a therapeutic protein,wherein said heterologous gene is expressed within a cell infected withsaid adenovirus. In a preferred embodiment, the therapeutic protein isselected from the group consisting of cytokines and chemokines,antibodies, pro-drug converting enzymes, and immunoregulatory proteins.

The present invention provides methods for using the chimericadenoviruses of the invention for therapeutic purposes. In oneembodiment, the chimeric adenoviruses can be used to inhibit the growthof cancer cells. In a particular embodiment, a chimeric adenoviruscomprising SEQ ID NO: 1 is useful for inhibiting the growth of coloncancer cells.

In another embodiment, the adenoviruses of the invention are useful asvectors to deliver therapeutic proteins to cells.

The present invention provides a method for production of the chimericadenoviruses of the invention, wherein the method comprises

-   -   a) pooling of adenoviral serotypes representing adenoviral        subgroups B-F, thereby creating an adenoviral mixture;    -   b) passaging the pooled adenoviral mixture from step (a) on an        actively growing culture of tumor cells at a particle per cell        ratio high enough to encourage recombination between serotypes,        but not so high as to produce premature cell death;    -   c) harvesting the supernatant from step (b);    -   d) infecting a quiescent culture of tumor cells with the        supernatant harvested in step (c);    -   e) harvesting the cell culture supernatant from step (d) prior        to any sign of CPE;    -   f) infecting a quiescent culture of tumor cells with the        supernatant harvested in step (e); and    -   g) isolating the chimeric adenovirus from the supernatant        harvested in step (f) by plaque purification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Ad retention time profiles on a TMAE HPLC column. A) Retentionprofiles for the individual Ad serotypes that were used to generate theoriginal starting viral pool. B) Retention profiles of the passage 20pools derived from HT-29, Panc-1, MDA-231, and PC-3 cell lines,respectively.

FIG. 2. Cytolytic activity of the individual virus pools. A) HT-29, B)MDA-231, C) Panc-1 and D) PC-3 cells were infected with their respectiveviral pools at VP per cell ratios from 100 to 0.01. MTS assays wereperformed on differing days post infection (as indicated) dependent uponthe cell line. Each data point in the panel represents an assay done inquadruplicate and the results are expressed as the means ±SD. The paneldepicts one representative experiment and all viral pools were assayedat least three independent times on the target tumor cell line (FigureLegend: -●-Ad5; -□- initial viral pool; -▪- specific cell derived pool,passage 20).

FIG. 3. Cytolytic activity of ColoAd1 and Ad5 on human tumor cell lines.An MTS assay was performed on A) a broad panel of human tumor cell linesand B) on a panel of human colon cancer cell lines to determine itspotential potency specificity. The MTS assay was performed on differingdays dependent upon the cell line. Each panel is a representativeexperiment that has been repeated at least three times. Each data pointin the panel represents an assay done in quadruplicate and the resultsare expressed as the means ±SD (Figure Legend: -●- Ad5; -□- ColoAd1).

FIG. 4. Cytolytic activity of ColoAd1 and Ad5 on a panel of normalcells. HS-27, HUVEC and SAEC cells (primary fibroblast, endothelial, andepithelial cells, respectively) were infected with ColoAd1 and Ad5 at VPper cell ratios from 100 to 0.01. MTS assay was performed on differingdays post infection dependent upon the cell and each panel is arepresentative experiment that has been repeated at least three times.Each data point in the panel represents an assay done in quadruplicateand the results are expressed as the means ±SD (Figure Legend: -●- Ad5;-□- ColoAd1).

FIG. 5. Cytolytic activity of ColoAd1, Ad5 and ONYX-015 on primarynormal endothelial cells (HUVEC) and a colon tumor cell line (HT-29).Each panel is a representative experiment that has been repeated atleast three times. Each data point in the panel represents an assay donein quadruplicate and the results are expressed as the means ±SD (FigureLegend: -●- Ad5; -□- ColoAd1; -▪- Onyx-015).

FIG. 6. Cytolytic activity of ColoAd1, Ad11p and Ad5 on a normalepithelial cell line (SAEC) and a human colon cancer cell line (HT-29).Each panel is a representative experiment that has been repeated atleast three times. Each data point in the panel represents an assay donein quadruplicate and the results are expressed as the means ±SD (FigureLegend: -●- Ad5; -□- Ad11p; -▪- ColoAd1).

FIG. 7. Cytolytic activity of Recombinant Viruses. Recombinant virusesrepresenting four viral populations (Adp11, ColoAd1, left endAd11p/right end ColoAd1(ColoAd1.1) and left end ColoAd1/right end Ad11p(ColoAd1.2)) were constructed as described in Example 6. Cytolyticactivity of each population in HT29 cells was determined as previouslydescribed. (Figure Legend: -●- Ad5; -□- AAd11p; -▪- ColoAd1; -υ-ColoAd1.1;-▴- ColoAd1.2).

DETAILED DESCRIPTION OF THE INVENTION

All publications, including patents and patent applications, mentionedin this specification are herein incorporated by reference to the sameextent as if each individual publication was specifically andindividually indicated to be incorporated by reference in its entirety.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures described below are those wellknown and commonly employed in the art.

As used herein, the term “adenovirus”, “serotype” or “adenoviralserotype” refers to any of the 51 human adenoviral serotypes currentlyknown, or isolated in the future. See, for example, Strauss, “Adenovirusinfections in humans,” in The Adenoviruses, Ginsberg, ea., Plenum Press,New York, N.Y., pp. 451-596 (1984). These serotypes are classified inthe subgroups A-F (see, Shenk, “Adenoviridae: The Viruses and TheirReplication,” in Fields Virology, Vol. 2, Fourth Edition, Knipe, ea.,Lippincott Williams & Wilkins, pp. 2265-2267 (2001), as shown inTable 1. TABLE 1 SubGroup Adenoviral Serotype A 12, 18, 31 B 3, 7, 11,14, 16, 21, 34, 35, 51 C 1, 2, 5, 6 D 8-10, 13, 15, 17, 19, 20, 22-30,32, 33, 36-39, 42-49, 50 E 4 F 40, 41

As used herein, “chimeric adenovirus” refers to an adenovirus whosenucleic acid sequence is comprised of the nucleic acid sequences of atleast two of the adenoviral serotypes described above.

As used herein, “parent adenoviral serotype” refers to the adenoviralserotype which represents the serotype from which the majority of thegenome of the chimeric adenovirus is derived.

As used herein, the term “homologous recombination” refers to twonucleic acid molecules, each having homologous sequences, where the twonucleic acid molecules cross over or undergo recombination in the regionof homology.

As used herein, the term “potency” refers to the lytic potential of avirus and represents its ability to replicate, lyse, and spread. For thepurposes of the instant invention, potency is a value which compares thecytolytic activity of a given adenovirus of the invention to that of Ad5in the same cell line, i.e. potency=IC₅₀ of AdX/IC₅₀ of Ad5, where X isthe particular adenoviral serotype being examined and wherein thepotency of Ad5 is given a value of 1.

As used herein, the term “oncolytic virus” refers to a virus thatpreferentially kills cancer cells as compared with normal cells.

As used herein, the term “therapeutic index” or “therapeutic window”refers to a number indicating the oncolytic potential of a givenadenovirus and is determined by dividing the potency of the adenovirusin a cancer cell line by the potency of the same adenovirus in a normal(i.e. non-cancerous) cell line.

As used herein, the term “modified” refers to a molecule with anucleotide or amino acid sequence differing from a naturally-occurring,e.g. a wild-type nucleotide or amino acid sequence. A modified moleculecan retain the function or activity of a wild-type molecule, i.e. amodified adenovirus may retain its oncolytic activity. Modificationsinclude mutations to nucleic acids as described below.

As used herein, “mutation” with reference to a polynucleotide orpolypeptide, refers to a naturally-occurring, synthetic, recombinant, orchemical change or difference to the primary, secondary, or tertiarystructure of a polynucleotide or polypeptide, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide). Mutations include such changesas, for example, deletions, insertions, or substitutions.Polynucleotides and polypeptides having such mutations can be isolatedor generated using methods well known in the art.

As used herein, “deletion” is defined as a change in eitherpolynucleotide or amino acid sequences in which one or morepolynucleotides or amino acid residues, respectively, are absent.

As used herein, “insertion” or “addition” is that change in apolynucleotide or amino acid sequence which has resulted in the additionof one or more polynucleotides or amino acid residues, respectively, ascompared to the naturally occurring polynucleotide or amino acidsequence.

As used herein, “substitution” results from the replacement of one ormore polynucleotides or amino acids by different polynucleotides oramino acids, respectively.

As used herein, the term “adenoviral derivative” refers to an adenovirusof the invention that has been modified such that an addition, deletionor substitution has been made to or in the viral genome, such that theresulting adenoviral derivative exhibits a potency and/or therapeuticindex greater than that of the parent adenovirus, or in some other wayis more therapeutically useful (i.e., less immunogenic, improvedclearance profile). For example, a derivative of an adenovirus of theinvention may have a deletion in one of the early genes of the viralgenome, including, but not limited to, the E1A or E2B region of theviral genome.

As used herein, “variant” with reference to a polynucleotide orpolypeptide, refers to a polynucleotide or polypeptide that may vary inprimary, secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide). For example, the amino acid ornucleic acid sequence may contain a mutation or modification thatdiffers from a reference amino acid or nucleic acid sequence. In someembodiments, an adenoviral variant may be a different isoform orpolymorphism. Variants can be naturally-occurring, synthetic,recombinant, or chemically modified polynucleotides or polypeptidesisolated or generated using methods well known in the art. Changes inthe polynucleotide sequence of the variant may be silent. That is, theymay not alter the amino acids encoded by the polynucleotide. Wherealterations are limited to silent changes of this type, a variant willencode a polypeptide with the same amino acid sequence as the reference.Alternatively, such changes in the polynucleotide sequence of thevariant may alter the amino acid sequence of a polypeptide encoded bythe reference polynucleotide, resulting in conservative ornon-conservative amino acid changes, as described below. Suchpolynucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. Various codon substitutions, such as thesilent changes that produce various restriction sites, may be introducedto optimize cloning into a plasmid or viral vector or expression in aparticular prokaryotic or eukaryotic system.

As used herein, an “adenoviral variant” refers to an adenovirus whosepolynucleotide sequence differs from a reference polynucleotide, e.g. awild-type adenovirus, as described above. The differences are limited sothat the polynucleotide sequences of the parent and the variant aresimilar overall and, in most regions, identical. As used herein, a firstnucleotide or amino acid sequence is said to be “similar” to a secondsequence when a comparison of the two sequences shows that they have fewsequence differences (i.e., the first and second sequences are nearlyidentical). As used herein, the polynucleotide sequence differencespresent between the adenoviral variant and the reference adenovirus donot result in a difference in the potency and/or therapeutic index.

As used herein, the term “conservative” refers to substitution of anamino acid residue for a different amino acid residue that has similarchemical properties. Conservative amino acid substitutions includereplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, or a threonine with a serine. Insertions or deletions aretypically in the range of about 1 to 5 amino acids.

As used herein, the term “nonconservative” refers to substituting anamino acid residue for a different amino acid residue that has differentchemical properties. The nonconservative substitutions include, but arenot limited to aspartic acid (D) being replaced with glycine (G);asparagine (N) being replaced with lysine (K); or alanine (A) beingreplaced with arginine (R).

The single-letter codes for amino acid residues include the following:A=alanine, R=arginine, N=asparagine, D=aspartic acid, C=cysteine,Q=Glutamine, E=Glutamic acid, G=glycine, H=histidine, I=isoleucine,L=leucine, K=lysine, M=methionine, F=phenylalanine, P=proline, S=serine,T=threonine, W=tryptophan, Y=tyrosine, V=valine.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Even the common modifications that occur naturally inpolypeptides are too numerous to list exhaustively here, but they arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. Among the known modifications which may be presentin polypeptides of the present invention are, to name an illustrativefew, acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a polynucleotide or polynucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, I. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W.H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12,1983; Seifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationalevents, including natural processing events and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationalnatural processes and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentto the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

As used herein, the following terms are used to describe the sequencerelationships between two or more polynucleotide or amino acidsequences: “reference sequence”, “comparison window”, “sequenceidentity”, “percentage of sequence identity”, “substantial identity”,“similarity”, and “homologous”. A “reference sequence” is a definedsequence used as a basis for a sequence comparison; a reference sequencemay be a subset of a larger sequence, for example, as a segment of afull-length cDNA or gene sequence given in a sequence listing or maycomprise a complete cDNA or gene sequence. Generally, a referencesequence is at least 18 nucleotides or 6 amino acids in length,frequently at least 24 nucleotides or 8 amino acids in length, and oftenat least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted, for example, by the local homology algorithm of Smith andWaterman, Adv. Appl. Math. 2:482 (1981), by the homology alignmentalgorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by thesearch for similarity method of Pearson and Lipman, Proc. Natl. Acad.Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, (Genetics Computer Group, 575 Science Dr.,Madison, Wis.), VectorNTI from Informatix, Geneworks, or MacVectorsoftware packages), or by inspection, and the best alignment (i. e.,resulting in the highest percentage of homology over the comparisonwindow) generated by the various methods is selected.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, U, or I) or residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide or amino acid sequence, wherein thepolynucleotide or amino acid comprises a sequence that has at least 85percent sequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 18nucleotide (6 amino acid) positions, frequently over a window of atleast 24-48 nucleotide (8-16 amino acid) positions, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to the sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the comparisonwindow. The reference sequence may be a subset of a larger sequence. Theterm “similarity”, when used to describe a polypeptide, is determined bycomparing the amino acid sequence and the conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.The term “homologous”, when used to describe a polynucleotide, indicatesthat two polynucleotides, or designated sequences thereof, whenoptimally aligned and compared, are identical, with appropriatenucleotide insertions or deletions, in at least 70% of the nucleotides,usually from about 75% to 99%, and more preferably at least about 98 to99% of the nucleotides.

As used herein, “homologous”,when used to describe a polynucleotide,indicates that two polynucleotides, or designated sequences thereof,when optimally aligned and compared, are identical, with appropriatenucleotide insertions or deletions, in at least 70% of the nucleotides,usually from about 75% to 99%, and more preferably at least about 98 to99% of the nucleotides.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure wherein specific pieces of DNA are amplified as described inU.S. Pat. No. 4,683,195. Generally, sequence information from the endsof the polypeptide fragment of interest or beyond needs to be available,such that oligonucleotide primers can be designed; these primers willpoint towards one another, and will be identical or similar in sequenceto opposite strands of the template to be amplified. The 5′ terminalnucleotides of the two primers will coincide with the ends of theamplified material. PCR can be used to amplify specific DNA sequencesfrom total genomic DNA, cDNA transcribed from total cellular RNA,plasmid sequences, etc. (See generally Mullis et al., Cold Spring HarborSymp. Quant. Biol., 51: 263, 1987; Erlich, ed., PCR Technology, StocktonPress, NY, 1989).

As used herein, “stringency” typically occurs in a range from aboutT_(m) (melting temperature)−5° C. (5° below the T_(m) of the probe) toabout 20° C. to 25° C. below T_(m). As will be understood by those ofskill in the art, a stringent hybridization can be used to identify ordetect identical polynucleotide sequences or to identify or detectsimilar or related polynucleotide sequences. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.

As used herein, “hybridization” as used herein, shall include “anyprocess by which a polynucleotide strand joins with a complementarystrand through base pairing” (Coombs, J., Dictionary of Biotechnology,Stockton Press, New York, N.Y., 1994).

As used herein, the term “therapeutically effective dose” or “effectiveamount” refers to that amount of adenovirus which ameliorates thesymptoms or conditions of a disease. A dose is considered atherapeutically effective dose in the treatment of cancer or itsmetastasis when tumor or metastatic growth is slowed or stopped, or thetumor or metastasis is found to shrink in size, so as to lead to anextension in life-span for the subject.

ADENOVIRUSES OF THE INVENTION

The present invention provides chimeric adenoviruses, or variants orderivatives thereof, having a genome in which the nucleotide sequence ofthe E2B region of the chimeric adenovirus comprises nucleic acidsequences derived from at least two adenoviral serotypes, whichserotypes are each selected from the adenoviral subgroups B, C, D, E andF and are distinct from each other. A chimeric adenovirus of theinvention is oncolytic and demonstrates an enhanced therapeutic indexfor a tumor cell.

Isolation of Chimeric Adenoviruses

The chimeric adenoviruses of the invention, or variants or derivativesthereof, can be produced using modification of a technique referred toas “bioselection”, in which an adenovirus with desired properties, suchas enhanced oncogenicity or cell type specificity, is generated throughthe use of genetic selection under controlled conditions (Yan et al.(2003) J. Virol. 77:2640-2650).

In the present invention, a mixture of adenoviruses of differingserotypes is pooled and is passaged, preferably at least twice, on asubconfluent culture of tumor cells at a particle per cell ratio highenough to encourage recombination between serotypes, but not so high asto produce premature cell death. A preferred particle per cell ratio isapproximately 500 particles per cell, and is easily determined by oneskilled in the art. As used herein, a “subconfluent culture” of cellsrefers to a monolayer or suspension culture in which the cells areactively growing. For cells grown as a monolayer, an example would be aculture where approximately 50% to 80% of the area available for cellgrowth is covered with cells. Preferred is a culture where approximately75% of the growth area is covered with cells.

In a preferred embodiment, the adenoviral mixture is one that includesadenoviral serotypes representative of the adenoviral subgroups B, C, D,E and F. Group A adenoviruses are not included in the mixture as theyare associated with tumor formation in rodents. Preferred tumor celllines useful in the bioselection process include, but are not limitedto, those derived from breast, colon, pancreas, lung and prostate. Someexamples of solid tumor cell lines useful for the “bioselective”passaging of the adenoviral mixture include, but are not limited to,MDA231, HT29, PAN-1 and PC-3 cells. Hemopoietic cell lines include, butare not limited to, the Raji and Daudi B-lymphoid cells, K562erythroblastoid cells, U937 myeloid cells, and HSB2 T-lymphoid cells.

Adenoviruses produced during these initial passages are used to infectquiescent tumor cells at a particle to cell ratio low enough to permitthe infection of a cell by no more than one adenovirus. After up to 20passages under these conditions, the supernatant from the last passageis harvested prior to visible cytopathic effect (CPE, see FieldsVirology, Vol. 2, Fourth Edition, Knipe, ea., Lippincott Williams &Wilkins, pp. 135-136) to increase selection of highly potent viruses.The harvested supernatant can be concentrated by techniques well knownto those skilled in the art. A preferred method for attaining quiescentcells, i.e. ones in which active cell growth has stopped, in a monolayerculture is to allow the culture to grow for 3 days following confluence,where confluence means that the entire area available for cell growth isoccupied (covered with cells). Similarly, suspension cultures can begrown to densities characterized by the absence of active cell growth.

The serotype profile of the concentrated supernatant, which contains thebioselected adenoviral pool, can be examined by measuring the retentiontimes of the harvested viral pool on an anion exchange column, wheredifferent adenoviral serotypes are known to have characteristicretention times (Blanche et al. (2000) Gene Therapy 7:1055-1062); seeExample 3, FIGS. 1A and B. Adenoviruses of the invention can be isolatedfrom the concentrated supernatant by dilution and plaque purification,or other techniques well know in the art, and grown for furthercharacterization. Techniques well known in the art are used to determinethe sequence of the isolated chimeric adenoviruses (see Example 5).

An example of a chimeric adenovirus of the invention is the chimericadenovirus ColoAd1, which was isolated using HT29 colon cells in thebioselection process. ColoAd1 has the nucleic acid sequence of SEQ IDNO: 1. The majority of the nucleotide sequence of ColoAd1 is identicalto the nucleotide sequence of the Ad11 serotype (SEQ ID NO: 2) (Stone etal. (2003) Virology 309:152-165; Mei et al. (2003) J. Gen. Virology84:2061-2071). There are two deletions in the ColoAd1 nucleotidesequence as compared with Ad11, one 2444 base pairs in length within theE3 transcription unit region of the genome (base pairs 27979 to 30423 ofSEQ ID NO: 2) and a second, smaller deletion, 25 base pairs in length(base pairs 33164 to 33189 of SEQ ID NO: 2), within the E4orf4 gene. TheE2B transcription unit region (SEQ ID NO: 3) of ColoAd1, which encodesthe adenoviral proteins DNA polymerase and terminal protein, is locatedbetween base pairs 5067 and 10354 of SEQ ID NO: 1, and is an area ofhomologous recombination between the Ad11 and Ad3 serotypes. Within thisregion of ColoAd1, there are 198 base pair changes, as compared with thesequence of Ad11 (SEQ ID NO: 1). The changes result in stretches ofnucleotides within the E2B region of ColoAd1 which are homologous to thesequence within a portion of the E2B region of Ad3 (SEQ ID NO: 8), withthe longest stretch of homology between ColoAd1 and Ad3 being 414 bp inlength. The E2B region of ColoAd1 (SEQ ID NO: 3) confers enhancedpotency to the ColoAd1 adenovirus as compared to unmodified Ad11adenovirus (see Example 6; FIG. 7). In other embodiments, a chimericadenovirus of the invention can comprise nucleic acid sequences frommore than two adenoviral serotypes.

A chimeric adenovirus of the invention, or a variant or derivativethereof, can be evaluated for its selectivity in a specific tumor typeby examination of its lytic potential in a panel of tumor cells derivedfrom the same tissue upon which the adenoviral pool was initiallypassaged. For example, the chimeric adenovirus ColoAd1 (SEQ ID NO: 1),which was initially derived from an adenoviral pool passaged on HT-29colon tumor cell lines, was re-examined both in HT-29 cells and in apanel of other colon-derived tumor cells lines, including DLD-1, LS174T,LS1034, SW403, HCI116, SW48, and Colo320DM (see FIG. 3B). Any availablecolon tumor cell lines would be equally useful for such an evaluation.Isolated adenoviral clones from adenoviral pools selected on other tumorcell types can be similarly tested in a suitable tumor cell panel,including, but not limited to, prostate cell lines (e.g. DU145 and PC-3cell lines); pancreatic cell lines (e.g. the Panc-1 cell line); breasttumor cell lines (e.g. the MDA231 cell line) and ovarian cell lines(e.g. the OVCAR-3 cell line). Other available tumor cell lines areequally useful in isolating and identifying adenoviruses of theinvention.

The chimeric adenoviruses of the invention have an enhanced therapeuticindex as compared with the adenoviral serotypes from which it isderived. (see FIG. 6, which compares the cytolytic activity of thechimeric adenovirus ColoAd1 with Ad11p).

The invention also encompasses chimeric adenoviruses that areconstructed using recombinant techniques well-known to those skilled inthe art. Such chimeric adenoviruses comprise a region of nucleotidesequence derived from one adenoviral serotype which is incorporated byrecombinant techniques into the genome of a second adenoviral serotype.The incorporated sequence confers a property, e.g. tumor specificity orenhanced potency, to the parental adenoviral serotype. For example, theE2B region of ColoAd1 (SEQ ID NO: 3) can be incorporated into the genomeof Ad35 or Ad9.

Adenoviral Derivatives

The invention also encompasses a chimeric adenovirus of the inventionthat is modified to provide other therapeutically useful chimericadenoviruses. Modifications include, but are not limited to, thosedescribed below.

One modification is production of derivatives of the chimeric adenovirusof the invention substantially lacking the ability to bind p53, as aresult of a mutation in the adenoviral gene that encodes the E1B-55Kprotein. Such viruses generally have some, or all, of the E1B-55K regiondeleted. (see U.S. Pat. No. 5,677,178). U.S. Pat. No. 6,080,578describes, among other things, Ad5 mutants that have deletions in theregion of the E1B-55K protein that is responsible for binding p53.Another preferred modification to the chimeric adenoviruses of theinstant invention are mutations in the E1A region, as described in U.S.Pat. Nos. 5,801,029 and 5,972,706. These types of modifications providederivatives of the chimeric adenoviruses of the invention with greaterselectivity for tumor cells.

Another example of a modification encompassed by the invention is achimeric adenovirus which exhibits an enhanced degree of tissuespecificity due to placement of viral replication under the control of atissue specific promoter as described in U.S. Pat. No. 5,998,205.Replication of a chimeric adenovirus of the invention can also be putunder the control of an E2F responsive element as described in U.S.patent application Ser. No. 09/714,409. This modification affords aviral replication control mechanism based on the presence of E2F,resulting in enhanced tumor tissue specificity, and is distinct from thecontrol realized by a tissue specific promoter. In both of theseembodiments, the tissue specific promoter and the E2F responsive elementare operably linked to an adenoviral gene that is essential for thereplication of the adenovirus.

Another modification encompassed by the invention is use of a chimericadenovirus of the invention, e.g. ColoAd1, as the backbone forproduction of novel replication-deficient adenoviral vectors. Asdescribed in Lai et al. ((2002) DNA Cell Bio. 21:895-913), adenoviralvectors which are replication deficient can be used to deliver andexpress therapeutic genes. Both first generation (in which the E1 andE3-regions are deleted) and second generation (in which the E4 region isadditionally deleted) adenoviral vectors derived from the chimercadenoviruses of the invention are provided herein. Such vectors areeasily produced using techniques well known to those skilled in the art(see Imperiale and Kochanek (2004) Curr. Top. Microbiol. Immunol.273:335-357; Vogels et al. (2003) J. Virol. 77:8263-8271).

A further modification encompassed by the invention is the insertion ofa heterologous gene, useful as a marker or reporter for tracking theefficiency of viral infection. One embodiment of this type ofmodification is insertion of the thymidine kinase (TK) gene. Theexpression of TK within infected cells can be used to track the level ofvirus remaining in cells following viral infection, using radiolabeledsubtrates of the TK reaction (Sangro et al. (2002) Mol. Imaging Biol.4:27-33).

Methods for the construction of the modified chimeric adenoviruses aregenerally known in the art. See, Mittal, S. K. (1993) Virus Res.28:67-90 and Hermiston, T. et al. (1999) Methods in Molecular Medicine:Adenovirus Methods and Protocols, W. S. M. Wold, ed, Humana Press.Standard techniques are used for recombinant nucleic acid methods,polynucleotide synthesis, and microbial culture and transformation(e.g., electroporation, lipofection). Generally, enzymatic reactions andpurification steps are performed according to the manufacturersspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd. edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) which are provided throughout thisdocument. The nomenclature used herein and the laboratory procedures inanalytical chemistry, organic synthetic chemistry, and pharmaceuticalformulation described below are those well known and commonly employedin the art.

Determination of Therapeutic Potential

Chimeric adenoviruses of the invention, or variants or derivativesthereof, can be evaluated for their therapeutic utility by examinationof their lytic potential in tumor cells derived from tissues of interestas therapeutic targets. Tumor cell lines useful for testing suchadenoviruses include, but are not limited to, colon cell lines,including but not limited to, DLD-1, HCT116, HT29, LS1034 and SW48 celllines; prostate cell lines, including but not limited to, DU145 and PC-3cell lines; pancreatic cell lines, including but not limited to, thePanc-1 cell line; breast tumor cell lines, including but not limited to,the MDA231 cell line and ovarian cell lines, including but not limitedto, the OVCAR-3 cell line. Hemopoietic cell lines include, but are notlimited to, the Raji and Daudi B-lymphold cells, K562 erythroblastoidcells, U937 myeloid cells, and HSB2 T-lymphoid cells. Any other tumorcell lines that are available can be used in evaluating and identifyingadenoviruses of the invention for use in the treatment of neoplasia.

The cytolytic activity of adenoviruses of the invention can bedetermined in representative tumor cell lines and the data converted toa measurement of potency, with an adenovirus belonging to subgroup C,preferably Ad5, being used as a standard (i.e. given a potency of 1). Apreferred method for determining cytolytic activity is an MTS assay (seeExample 4, FIG. 2).

The therapeutic index of an adenovirus of the invention in a particulartumor cell line can be calculated by comparison of the potency of thegiven adenovirus in a tumor cell line with the potency of that sameadenovirus in a noncancerous cell line. Preferred non-cancerous celllines are SAEC cells, which are epithelial in origin, and HUVEC cellswhich are endothelial in origin (see FIG. 4). These two cell typesrepresent normal cells from which organs and vasculature, respectively,are derived, and are representative of likely sites of toxicity duringadenoviral therapy, depending on the mode of delivery of the adenovirus.However, practice of the invention is not limited to the use of thesecells, and other non-cancerous cell lines (e.g. B cells, T cells,macrophages, monocytes, fibroblasts) may also be used.

The chimeric adenoviruses of the invention can be further evaluated fortheir ability to target neoplastic cell growth (i.e. cancer) by theircapacity to reduce tumorigenesis or neoplastic cell burden in nude miceharboring a transplant of neoplastic cells, as compared to untreatedmice harboring an equivalent neoplastic cell burden (see Example 7).

Evaluation of the adenoviruses of the invention can also be performedusing primary human tumor explants (Lam et al. (2003) Cancer GeneTherapy; Grill et al. (2003) Mol. Therapy 6:609-614), which provide testconditions present in tumors that cannot normally be produced using thetumor xenograft studies.

Therapeutic Utility

The present invention provides for the use of chimeric adenoviruses ofthe invention for the inhibition of tumor cell growth, as well as forthe use of adenoviral vectors derived from these chimeric adenovirusesto deliver therapeutic proteins useful in the treatment of neoplasia andother disease states.

Pharmaceutical Compositions and Administration

The present invention also relates to pharmaceutical compositions whichcomprise the chimeric adenoviruses of the invention, including variantsand derivatives thereof, formulated for therapeutic administration to apatient. For therapeutic use, a sterile composition containing apharmacologically effective dosage of adenovirus is administered to ahuman patient or veterinary non-human patient for treatment, forexample, of a neoplastic condition. Generally, the composition willcomprise about 10¹¹ or more adenovirus particles in an aqueoussuspension. A pharmaceutically acceptable carrier or excipient is oftenemployed in such sterile compositions. A variety of aqueous solutionscan be used, e.g. water, buffered water, 0.4% saline, 0.3%-glycine andthe like. These solutions are sterile and generally free of particulatematter other than the desired adenoviral vector. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, e.g. sodium acetate,sodium chloride, potassium chloride, calcium chloride, sodium lactate,etc. Excipients which enhance infection of cells by adenovirus may beincluded. (see U.S. Pat. No. 6,392,069) Adenoviruses of the inventionmay also be delivered to neoplastic cells by liposome or immunoliposomedelivery; such delivery may be selectively targeted to neoplastic cellson the basis of a cell surface property present on the neoplastic cellpopulation (e.g., the presence of a cell surface protein which binds animmunoglobulin in an immunoliposome). Typically, an aqueous suspensioncontaining the virions are encapsulated in liposomes or immunoliposomes.For example, a suspension of adenovirus virions can be encapsulated inmicelles to form immunoliposomes by conventional methods (U.S. Pat. No.5,043,164, U.S. Pat. No. 4,957,735, U.S. Pat. No. 4,925,661; Connor andHuang, (1985) J. Cell Biol. 101: 581; Lasic D. D. (1992) Nature 355:279; Novel Drug Delivery (eds. Prescott and Nimmo, Wiley, N.Y., 1989);Reddy et al. (1992) J. Immunol. 148:1585). Immunoliposomes comprising anantibody that binds specifically to a cancer cell antigen (e.g., CALLA,CEA) present on the cancer cells of the individual may be used to targetvirions to those cells (Fisher (2001) Gene Therapy 8:341-348).

To further increase the efficacy of the adenoviruses of the invention,they may be modified to exhibit enhanced tropism for particular tumorcell types. For example, as shown in PCT/US98/04964, a protein on theexterior coat of an adenovirus may be modified to display a chemicalagent, preferably a polypeptide, that binds to a receptor present ontumor cells to a greater degree than normal cells. (See also, U.S. Pat.Nos. 5,770,442 and 5,712,136). The polypeptide can be an antibody, andpreferably is a single chain antibody.

Adenoviral Therapy

The adenoviruses of the invention, or pharmaceutical compositionsthereof, can be administered for therapeutic treatment of neoplasticdisease or cancer. In therapeutic applications, compositions areadministered to a patient already affected by the particular neoplasticdisease, in an amount sufficient to cure or at least partially arrestthe condition and its complications. An amount adequate to accomplishthis is defined as a “therapeutically effective dose” or “efficaciousdose”. Amounts effective for this use will depend upon the severity ofthe condition, the general state of the patient, and the route ofadministration.

For example, but not by way of limitation, a human patient or non-humanmammal having a solid or haemotologic neoplastic disease, (e.g.pancreatic, colon, ovarian, lung, or breast carcinoma, leukemia ormultiple myeloma) may be treated by administering a therapeuticallyeffective dosage of an appropriate adenovirus of the invention, i.e. onewhich has been shown to have an improved therapeutic index for thattissue type. For example, a preferred chimeric adenovirus for thetreatment of colon cancer would be the adenovirus ColoAd1 (SEQ ID NO:1). Suspensions of infectious adenovirus particles may be delivered toneoplastic tissue by various routes, including intravenous,intraperitoneal, intramuscular, subdermal, and topical. An adenovirussuspension containing about 10³ to 10¹² or more virion particles per mlmay be administered by infusion (e.g., into the peritoneal cavity fortreating ovarian cancer, into the portal vein for treatinghepatocarcinoma or liver metastases from other non-hepatic primarytumors) or other suitable route, including direct injection into a tumormass (e.g. a breast tumor), enema (e.g., colon cancer), or catheter(e.g., bladder cancer). Other routes of administration may be suitablefor carcinomas of other origins, i.e. inhalation as a mist (e.g., forpulmonary delivery to treat bronchogenic carcinoma, small-cell lungcarcinoma non-small cell lung carcinoma, lung adenocarcinoma orlaryngeal cancer) or direct application to a tumor site (e.g.,bronchogenic carcinoma, nasopharyngeal carcinoma, laryngeal carcinoma,cervical carcinoma).

Adenoviral therapy using the adenoviruses of the instant invention maybe combined with other antineoplastic protocols, such as conventionalchemotherapy or x-ray therapy to treat a particular cancer. Treatmentcan be concurrent or sequential. A preferred chemotherapeutic agent iscisplatin, and the preferred dose may be chosen by the practitionerbased on the nature of the cancer to be treated, and other factorsroutinely considered in administering cisplatin. Preferably, cisplatinwill be administered intravenously at a dose of 50-120 mg/m² over 3-6hours. More preferably it is administered intravenously at a dose of 80mg/m² over 4 hours. A second preferred chemotherapeutic agent is5-fluorouracil, which is often administered in combination withcisplatin. The preferred dose of 5-fluorouracil is 800-1200 mg/m² perday for 5 consecutive days.

Adenoviral therapy using the adenoviruses of the instant invention asadenoviral vectors may also be combined with other genes known to beuseful in viral based therapy. See U.S. Pat. No. 5,648,478. In suchcases, the chimeric adenovirus further comprises a heterologous genethat encodes a therapeutic protein, incorporated within the viralgenome, such that the heterologous gene is expressed within an infectedcell. A therapeutic protein, as used herein, refers to a protein thatwould be expected to provide some therapeutic benefit when expressed ina given cell.

In one embodiment, the heterologous gene is a pro-drug activator gene,such as cytosine deaminase (CD) (See, U.S. Pat. Nos. 5,631,236;5,358,866; and 5,677,178). In other embodiments, the heterologous geneis a known inducer of cell-death, e.g apoptin or adenoviral deathprotein (ADP), or a fusion protein, e.g. fusogenic membrane glycoprotein(Danen-Van Oorschot et al. (1997) Proc. Nat. Acad. Sci. 94:5843-5847;Tollefson et al.(1996) J. Virol. 70:2296-2306; Fu et al. (2003) Mol.Therapy 7: 48-754, 2003; Ahmed et al. (2003) Gene Therapy 10:1663-1671,Galanis et al. (2001) Human Gene Therapy 12(7): 811-821).

Further examples of heterologous genies, of fragments thereof, includethose that encode immunomodulatory proteins, such as cytokines orchemokines. Examples include interleukin 2, U.S. Pat. Nos. 4,738,927 or5,641,665; interleukin 7, U.S. Pat. Nos. 4,965,195 or 5,328,988; andinterleukin 12, U.S. Pat. No. 5,457,038; tumor necrosis factor alpha,U.S. Pat. Nos. 4,677,063 or 5,773,582; interferon gamma, U.S. Pat. Nos.4,727,138 or 4,762,791; or GM CSF, U.S. Pat. Nos. 5,393,870 or5,391,485, Mackensen et al. (1997) Cytokine Growth Factor Rev.8:119-128). Additional immunomodulatory proteins further includemacrophage inflammatory proteins, including MIP-3. Monocyte chemotaticprotein (MCP-3 alpha) may also be used; a preferred embodiment of aheterologous gene is a chimeric gene consisting of a gene that encodes aprotein that traverses cell membranes, for example, VP22 or TAT, fusedto a gene that encodes a protein that is preferably toxic to cancer butnot normal cells.

The chimeric adenoviruses of the invention can also be used as vectorsto deliver genes encoding therapeutically useful RNA molecules, i.e.siRNA (Dorsett and Tuschl (2004) Nature Rev Drug Disc 3:318-329).

In some cases, genes can be incorporated into a chimeric adenovirus ofthe invention to further enhance the ability of the oncolytic virus toeradicate the tumor, although not having any direct impact on the tumoritself—these include genes encoding proteins that compromise MHC class Ipresentation (Hewitt et al. (2003) Immunology 110: 163-169), blockcomplement, inhibit IFNs and IFN-induced mechanisms, chemokines andcytokines, NK cell based killing (Orange et al., (2002) Nature Immunol.3: 1006-1012; Mireille et al. (2002) Immunogenetics 54: 527-542; Alcami(2003) Nature Rev. Immunol. 3: 36-50; down regulate the immune response(e.g. IL-10, TGF-Beta, Khong and Restifo (2002) Nature Immunol. 3:999-1005; 2002) and metalloproteases which can breakdown theextracelluar matrix and enhance spread of the virus within the tumor(Bosman and Stamenkovic (2003) J. Pathol. 2000: 423-428; Visse andNagase (2003) Circulation Res. 92: 827-839).

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

The present invention is further described by the following examples,which are illustrative of specific embodiments of the invention, andvarious uses thereof. These exemplifications, which illustrating certainspecific aspects of the invention, do not portray the limitations orcircumscribe the scope of the disclosed invention.

Unless otherwise Indicated, the practice of the present Inventionemploys conventional techniques of cell culture, molecular biology,microbiology, recombinant DNA manipulation, immunology science, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, e.g. Cell Biology: a Laboratory Handbook: J. Celis(Ed).Academic Press. N.Y. (1996); Graham, F. L. and Prevec, L.Adenovirus-based expression vectors and recombinant vaccines. In:Vaccines: New Approaches to Immunological Problems. R. W. Ellis (ed)Butterworth. Pp 363-390; Grahan and Prevec Manipulation of adenovirusvectors. In: Methods in Molecular Biology, Vol. 7: Gene Transfer andExpression Techniques. E. J. Murray and J. M. Walker (eds) Humana PressInc., Clifton, N. J. pp 109-128, 1991; Sambrook et al. (1989), MolecularCloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress; Sambrook et al. (1989), and Ausubel et al. (1995), ShortProtocols in Molecular Biology, John Wiley and Sons.

EXAMPLES

Methods

Standard techniques are used for recombinant nucleic acid methods,polynucleotide synthesis, and microbial culture and transformation(e.g., electroporation, lipofection). Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd. edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) which are provided throughout thisdocument. The nomenclature used herein and the laboratory procedures inanalytical chemistry, organic synthetic chemistry, and pharmaceuticalformulation and delivery, and treatment of patients. Methods for theconstruction of adenoviral mutants are generally known in the art. See,Mittal, S. K., Virus Res.,1993, vol: 28, pages 67-90; and Hermiston, T.et al., Methods in Molecular Medicine: Adenovirus Methods and Protocols,W. S. M. Wold, ed, Humana Press, 1999. Further, the adenovirus 5 genomeis registered as Genbank 10 accession #M73260, and the virus isavailable from the American Type Culture Collection, Rockville, Md., U.S. A., under accession number VR-5.

Viruses and Cell Lines

The Ad serotypes Ad3 (GB strain), Ad4 (RI-67 strain), Ad5 (Adenoid 75strain), Ad9 (Hicks strain), Ad11p (Slobitski strain), Ad16 (Ch. 79strain) and all the cell lines, with the exception of the following wereall purchased from the ATCC: MDA231-mt1 (a derivative isolated by Dr.Deb Zajchowski from a rapidly growing subcutaneous implanted xenograftof MDA231 cells) and Panc1-sct (derived by Dr. Sandra Biroc from arapidly growing subcutaneous implanted xenograft of Panc1 cells), HUVEC(Vec Technologies, Rensselaer, N.Y.), and SAEC (Clonetics, Walkersville,Md.). Ad40 was a kind gift from Dr. William S. M. Wold at St. LouisUniversity.

Example 1 Viral Purification and Quantitation

Viral stocks were propagated on 293 cells and purified on CsCl gradients(Hawkins et al., 2001). The method used to quantitate viral particles isbased on that of Shabram et al. (1997) Human Gene Therapy 8:453-465,with the exception that the anion-exchanger TMAE Fractogel was usedinstead of Resource Q. In brief, a 1.25 ml column was packed withFractogel EMD TMAE-650 (S) (catalog # 116887-7 EM Science, Gibbstown,N.J. 08027). HPLC separation was performed on an Agilent HP 1100 HPLCusing the following conditions: Buffer A=50 mM HEPES, pH 7.5; BufferB=1.0 M NaCl in Buffer A; flow rate of 1 ml per minute. After columnequilibration for not less than 30 minutes in Buffer A, approximately10⁹-10¹¹ viral particles of sample were loaded onto the column in 10-100ul volume, followed by 4 column volumes of Buffer A. A linear gradientextending over 16 column volumes and ending in 100% Buffer B wasapplied.

The column effluent was monitored at A260 and A280 nm, peak areascalculated, and the 260 to 280 nm ratio determined. Viral peaks wereidentified as those peaks having a A260/A280 ratio close to 1.33. Avirus standard was included with each sample series. The number of viralparticles per ml of the standard had been determined using the method ofLehmberg et al. (1999) J. Chrom. B, 732:411-423}. In the viralconcentration range used, the A260 nm peak area of each sample isdirectly proportional to the number of viral particles in the sample.The number of viral particles per ml in each test sample was calculatedby multiplying the known number of viral particles per ml in thestandard by the ratio of the A260 nm viral peak area of the sample tothe A260 nm viral peak area of the standard.

The column was regenerated after each sample gradient by washing withtwo column volumes of 0.5 N NaOH followed by two column volumes of 100%Buffer A, 3 column volumes of 100% Buffer B, and then 4 column volumesof 100% Buffer A.

Example 2 Bioselection

Viral serotypes representing subgroups Ads B-F were pooled and passagedon sub-confluent cultures of the target tumor cell lines at a highparticle-per-cell ratio for two rounds to invite recombination to occurbetween serotypes. Supernatant (1.0, 0.1 0.01, 0.001 ml) from the secondround of the high viral particle-per-cell infection, subconfluentcultures, was then used to infect a series of over-confluent T-75 tissueculture flasks of target tumor cell lines PC-3, HT-29, Panc-1 andMDA-231. To achieve over-confluency, each cell line was seeded at splitratios that allowed that cell line to reach confluency between 24 and 40hours post seeding, and the cells were allowed to grow a total of 72hours post seeding prior to infection. This was done to maximize theconfluency of the cells to mimic growth conditions in human solidtumors.

Cell culture supernatant was harvested from the first flask in the10-fold dilution series that did not show any sign of CPE at day 3 or 4post-infection (in the case of HT-29 and PC-3, this was modified forpassages 10-20 to harvest of the second flask, i.e. harvest 100-foldbelow the dilution in which CPE were detectable by day 3post-infection). Each harvest served as the starting material for thesuccessive passage of the virus. This process was repeated until theviral pool achieved 20 bioselective passages.

Individual viruses from each bioselected pool were isolated by tworounds of plaque purification on A549 cells using standard methods(Tollefson, A., Hermiston, T. W., and Wold, W. S. M.; “Preparation andTitration of CsCl-banded Adenovirus Stock” in Adenovirus Methods andProtocols, Humana Press, 1999, pp 1-10, W. S. M. Wold, Ed). In brief,dilutions of the supernatant harvested from the 20^(th) passage on eachtarget tumor line were used to infect A549 cells in a standard plaqueassay. Well-individuated plaques were harvested, and the same plaqueassay method was used to generate a second round of individual plaquesfrom these harvests. Well isolated plaques from the second round ofplaque purification were deemed pure, infected cultures were preparedusing these purified plaques, and the oncolytic potency of these culturesupernatants determined by MTS assay as described.

Example 3 Serotype Characterization

The parental adenoviral serotypes comprising the viral pools or theisolated ColoAd1 adenovirus were identified using anion-exchangechromatography similar to that described in Shabram et al. (1997) HumanGene Therapy 8:453:465, with the exception that the anion-exchanger TMAEFractogel media (EM Industries, Gibbstown, N.J.) was used instead ofResource Q. as described in Example 1 (see FIG. 1).

Adenovirus type 5 eluted at approximately 60% Buffer B during thegradient. The other serotypes (3, 4, 9, 11 p, 16, 35, and 40) eacheluted at a characteristic retention time consistent with the retentiontimes on Q Sepharose XL published by Blanche et al. (2000) Gene Therapy7:1055-1062.

Example 4 Cytolytic Assay

The viral lytic capacity was measured by using a modification of the MTTassay (Shen et al., 2001). Briefly, the MTS assay (Promega, CellTiter96^((R)) Aqueous Non-Radioactive Cell Proliferation Assay) was used inplace of the MTT assay since conversion of MTS by cells Into aqueous,soluble formazan reduces time and eliminates the use of a volatileorganic solvent associated with the MTT assay.

To perform the assay, cells were seeded at a defined density for eachtumor cell line that generated a confluent monolayer within 24 hr. Thesedensely seeded cells were allowed to grow for 2 additional days prior toexposure to the test virus(es). Infections of both tumor and primarynormal cells were carried out in quadruplicate with serial three folddilutions of the viruses starting at a particle per cell ratio of 100and ending at a particle per cell ratio of 0.005. Infected cells wereincubated at 37° C. and the MTS assay was performed at the time pointsindicated for the individual primary cells or tumor cell lines.Mock-infected cells served as negative controls and established the 100%survival point for the given assay.

Example 5 DNA Sequencing

DNA sequencing of the Ad11p (SEQ ID NO: 2) and ColoAd1 (SEQ ID NO: 1)genomic DNAs was performed as follows. Briefly, purified adenovirus DNAfrom ColoAd1 and Ad11p was partially digested with the restrictionendonuclease Sau3A1 and shotgun cloned into the plasmid vectorpBluescript II (Stratagene, La Jolla, Calif.). Positive clones werepropagated and sequenced using the primers M13R and KS (Stratagene, LaJolla, Calif.). Individual sequence reactions were trimmed, edited andassembled using Sequencher^(tm) (Gene Codes Corp., Ann Arbor, Mich.).Gaps in coverage were amplified with custom oligonucleotide primers andsequenced. The ends of the viral genomes were sequenced directly off theadenoviral DNA. In all, each genome was sequenced at 3×+ coverage and431 bases at 2× coverage.

To determine the origin of the ColoAd1 E2B region, two primer sets weregenerated, one to the E2B pTP gene (bp9115, 5′GGGAGTTTCGCGCGGACACGG3′(SEQ ID NO: 4) and bp 9350, 5′GCGCCGCCGCCGCGGAGAGGT3′ (SEQ ID NO: 5))and one to the DNA polymerase gene (bp 7520 5′CGAGAGCCCATTCGTGCAGGTGAG3′(SEQ ID NO: 6) and bp 7982, 5′GCTGCGACTACTGCGGCCGTCTGT3′ (SEQ ID NO: 7)and used to PCR isolate DNA fragments from the various serotypes(Ad3,4,5,9,11 p,16 and 40) using reagents from the Advantage 2 PCR kit(Clonetics, Walkersville, Md.; Cat #K1910-Y) and run on a PTC-200thermocycler from MJ Research (Watertown, Mass.). These fragments weresubsequently sequenced along with the DNA sequence of Ad3 using dyeterminator sequencing on as ABI 3100 genetic analyzer.

The E2B region of Ad3 was sequenced using isolated Ad3 DNA andoverlapping primers.

Sequence information was analyzed using the Vector NTI program(Informatix).

Example 6 Construction of Recombinant Viruses

Genomic DNAs of Ad11p (SEQ ID NO: 2) and ColoAd1 (SEQ ID NO: 1) werepurified from CsCl gradient-banded virus particles. The genomic DNAswere digested with Pacl which cuts each only once within the viralgenome. The Pac1 cut occurs at base 18141 on ColoAd1 nucleotide sequence(SEQ ID NO: 1) and at base 18140 on the Ad11 nucleotide sequence (SEQ IDNO: 2). Digested DNAs were mixed in equal amounts and ligated in thepresence of T4 DNA ligase at 16° C. overnight. This ligation mixture wastransfected into A549 cells using the CaPO₄ transfection kit fromInvitrogen, Carlsbad, Calif. (Cat #K2780-01). Isolated plaques werepicked and screened by restriction enzyme digestion and PCR analysis todistinguish the four viral populations (Ad11p, ColoAd1, left endAd11p/right end ColoAd1 (ColoAd1.1) and left end ColoAd1/right endAd11p(ColoAd1.2)).

The viral lytic capacity of each population was determined in severalcell lines, including HT29 and HUVEC cell lines, as described in Example3. The results demonstrated the order of potency, from least potent tomost potent, as Ad11p, ColoAd1.2, ColoAd1.1, ColoAd1 (see FIG. 7 for theresults in HT29 cells).

Also constructed were chimeric adenoviruses pCJ144 and pCJ146, whichcontain the full-length ColoAd1 genome in which the wild-type Ad11p E3and E4 region, respectively, has been restored. These modifications wereintroduced by homologous recombination into BJ5183 E. Coli (Chartier etal. (1996) J. Virol. 70:4805-4810). Both of these chimeric adenovirusesdemonstrated reduced lytic capacity in HT29 and HUVEC cells compared toColoAd1 or ColoAd1.2.

Example 7 In Vivo Efficacy of Adenovirus

In a typical human tumor xenograft nude mouse experiment, animals areinjected with 5×10⁶ cells subcutaneously into the hind flank of themouse. When the tumors reach 100-200 ul in size, they are injected withvehicle (PBS) or with virus at 2×10¹⁰ particles for five consecutivedays (1×10¹¹ particles total). A reduction in the size of the tumorwould be noted relative to the PBS control and additional controlviruses (Ad5, ONYX-015).

Example 8 ColoAd1 Selectivity on Primary Human Tissue Explants

Tissue specimens from colorectal tumors and adjacent normal tissuesremoved during surgery were placed in culture media and infected withequal numbers of either ColoAd1 or Ad5 viruses. Culture supernatantswere collected at 24 hours post infection and the number of virusparticles produced was determined. ColoAd1 produced more virus particlesper input particle than Ad5 on tumor tissue, while it produced fewerparticles per input particle than Ad5 on normal tissue.

1. A recombinant chimeric adenovirus, or a variant or derivativethereof, having a genome comprising an E2B region wherein said E2Bregion comprises a nucleic acid sequence derived from a first adenoviralserotype and a nucleic acid sequence derived from a second adenoviralserotype; wherein said first and second serotypes are each selected fromthe adenoviral subgroups B, C, D, E, or F and are distinct from eachother; and wherein said chimeric adenovirus is oncolytic anddemonstrates an enhanced therapeutic index for a tumor cell.
 2. Theadenovirus of claim 1 further comprising regions encoding fiber, hexon,and penton proteins, wherein the nucleic acid encoding the fiber, hexon,and penton proteins of said adenovirus are from the same adenoviralserotype.
 3. The adenovirus of claim 1 further comprising a modified E3region.
 4. The adenovirus of claim 1 further comprising a modified E4region.
 5. The adenovirus of claim 1, wherein said tumor cell is acolon, breast, pancreas, lung, prostate, ovarian, or hemopoietic tumorcell.
 6. The adenovirus of claim 5, wherein said tumor cell is a colontumor cell.
 7. The adenovirus of claim 1, wherein the nucleotidesequence of the E2B region of said adenovirus comprises SEQ ID NO: 3, ora portion thereof.
 8. The adenovirus of claim 1, wherein the nucleotidesequence of said adenovirus comprises SEQ ID NO:
 1. 9. A recombinantchimeric adenovirts, or a variant or derivative thereof, having a genomecomprising an E2B region wherein said E2B region comprises a nucleicacid sequence derived from a first adenoviral serotype and a nucleicacid sequence derived from a second adenoviral serotype; wherein saidfirst and second adenoviral serotypes are each selected from theadenoviral subgroups B, C, D, E, or F and are distinct from each other;wherein said chimeric adenovirus is oncolytic and demonstrates anenhanced therapeutic index for a tumor cell; and wherein said chimericadenovirus has been rendered replication deficient through deletion ofone or more adenoviral regions encoding proteins involved in adenoviralreplication selected from the group consisting of E1, E2, E3 or E4. 10.The replication deficient adenovirus of claim 9, wherein the E1 and E3regions have been deleted.
 11. The replication deficient adenovirus ofclaim 10, further comprising a deletion of the E4 region.
 12. Theadenovirus of claim 1 or claim 9, further comprising a heterologousgene, wherein said heterologous gene is expressed within a cell infectedwith said adenovirus.
 13. The adenovirus of claim 12, wherein saidheterologous gene is thymidine kinase.
 14. The adenovirus of claim 12,wherein said heterologous gene encodes a therapeutic protein selectedfrom the group consisting of cytokines and chemokines, antibodies,pro-drug converting enzymes, and immunoregulatory proteins.
 15. A methodof inhibiting growth of a cancer cell, comprising infecting said cancercell with the adenovirus of claim
 1. 16. The method of claim 15, whereinsaid cancer cell is a colon cancer cell.
 17. The method of claim 16,wherein the nucleotide sequence of said adenovirus comprises SEQ IDNO:
 1. 18. A method of delivering a therapeutic protein to a cell,comprising Infecting the cell with the adenovirus of claim
 14. 19. Amethod for isolating the adenovirus of claim 1, wherein said methodcomprises a) pooling of adenoviral serotypes representing adenoviralsubgroups B-F, thereby creating an adenoviral mixture; b) passaging thepooled adenoviral mixture from step (a) on an actively growing cultureof tumor cells at a particle per cell ratio high enough to encouragerecombination between serotypes, but not so high as to produce prematurecell death; c) harvesting the supernatant from step (b); d) infecting aquiescent culture of tumor cells with the supernatant harvested in step(c); e) harvesting the cell culture supernatant from step (d) prior toany sign of CPE; f infecting a quiescent culture of tumor cells with thesupernatant harvested in step (e); and g) isolating the virus of claim 1from the supernatant harvested in step (f) by plaque purification. 20.The method of claim 19, wherein step (b) is performed twice beforeharvesting the supernatant in step (c).
 21. The method of claim 19,wherein steps (e) and (e are repeated up to 20 times prior to step (g).22. The method of claim 19, wherein a second round of plaquepurification is performed following step (g).
 23. The method of claim19, wherein the tumor cell is a colon, breast, pancreas, lung, prostate,ovarian, or hemopoietic tumor cell.
 24. A recombinant chimericadenovirus, or a variant or derivative thereof, having a genomecomprising an E2B region wherein said E2B region comprises a nucleicacid sequence derived from a first adenoviral serotype and a nucleicacid sequence derived from a second adenoviral serotype; wherein saidfirst and second adenoviral serotypes are each selected from theadenoviral subgroups B, C, D, E, or F and are distinct from each other;and wherein said chimeric adenovirus is oncolytic and demonstrates anenhanced therapeutic index for a tumor cell; produced by the method ofa) pooling of adenoviral serotypes representing adenoviral subgroupsB-F, thereby creating an adenoviral mixture; b) passaging the pooledadenoviral mixture from step (a) on an actively growing culture of tumorcells at a particle per cell ratio high enough to encouragerecombination between serotypes, but not so high as to produce prematurecell death; c) harvesting the supernatant from step (b); d) infecting aquiescent culture of tumor cells with the supernatant harvested in step(c); e) harvesting the cell culture supernatant from step (d) prior toany sign of CPE; f) infecting a quiescent culture of tumor cells withthe supernatant harvested in step (e); and g) isolating said chimericadenovirus from the supernatant harvested in step (f) by plaquepurification.
 25. The method of claim 24, wherein step (b) is performedtwice before harvesting of the supernatant in step (c).
 26. The methodof claim 24, wherein steps (e) and (e are repeated up to 20 times priorto step (g).
 27. The method of claim 24, wherein a second round ofplaque purification is performed following step (g).